Ozone treatment for elimination of pathogens

ABSTRACT

The present disclosure relates to the ozone treatment of plants to reduce or eliminate pesticides, mold, yeast, or other pathogens. In an embodiment, the plant is exposed to ozone at a concentration of 150 to 250 ppm for between 15 and 65 minutes. The exposure of the plant to ozone reduces or eliminates pesticides, mold, yeast or other pathogens with little to no negative effects on the plants potency, flavor profile, fragrance, and weight.

RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/496,912filed Apr. 25, 2017, which claims priority to U.S. Provisional PatentApplication Ser. No. 62/327,651 filed Apr. 26, 2016, both of which arefully incorporated herein by reference.

INTRODUCTION

The term pesticide covers a wide range of compounds includinginsecticides, fungicides, herbicides, rodenticides, molluscicides,nematicides, plant growth regulators and others. Pesticides aresubstances that are used to repel, kill or control animals(insecticides) or plants (herbicides) that are considered to be pests.Currently, the primary method of controlling such pests is through theapplication of pesticides, which often contain synthetic chemicalcompounds.

The introduction of synthetic insecticides—organo-phosphate insecticidesin the 1960s, carbamates in the 1970s, prethroids in the 1980s and theintroduction of herbicides and fungicides in the 1970s and 1980scontributed greatly to pest control and agricultural output. Ideally apesticide must be lethal to the targeted pests, but not to non-targetspecies, including animals and humans. Unfortunately, this is often notthe case.

Despite their agricultural and economic benefits, pesticides can havenegative impacts on human health. Many conventional pesticides aresynthetic materials that kill or inactivate a pest directly. Short-termexposure to a large amount of certain pesticides can result in seriouslong-term health detriments and death. Exposure to large amounts ofpesticides is usually more likely for people, such as farmers, who mayfrequently touch and/or breathe in pesticides. Studies have linked theeffects of long-term exposure to small amounts of pesticides to avariety of chronic health conditions such as diabetes, cancer andneurological defects, among others.

Studies have shown preliminary evidence that chronic, low-dose exposureto pesticides increases the risk of cognitive impairments and diseasessuch as Alzheimer's and Parkinson's later in life. A study of 50pesticides and more than 30,000 licensed pesticide applicators linkedexposure of seven pesticides that contain chlorinated compounds(including two herbicides, two organophosphate insecticides and twoorganochlorines, to increased risk of diabetes). Exposure to pesticideshas also been associated with increased infertility in women anddevelopmental problems in children.

The most widely used herbicide in the world, glyphosate, is employed inmass quantities in agriculture around the world. Although glyphosate isthought to be less toxic than many other traditional herbicides, theWorld Health Organization has announced that it is a probablecarcinogen.

In addition to linking herbicides to cancer, plants are known to developresistance to herbicides over time. Weeds that have developed resistanceto one herbicide may require that higher amounts of that herbicides beapplied to them to result in sufficient weed suppression and may alsorequire treatment of additional herbicides in a “herbicide cocktail” tokeep them under control. Thus, herbicide-tolerant crops will be exposedto higher levels of herbicides as resistance to the most commonly usedpesticides increases.

Pesticides have been found to dramatically affect the environment.Pesticides can contaminate soil, water, turf and other vegetation.

Heavy treatment of soil with pesticides can cause populations ofbeneficial soil microorganisms to decline. Overuse of chemicalfertilizer and pesticides have deleterious effects on the soil organismsthat are similar to the effects seen by human overuse of antibiotics.Indiscriminate use of chemicals might be useful in the short term, butif used for prolonged periods they can reduce the amount of beneficialnutrient-synthesizing soil organisms to a point where nitrate levels insoil are not sufficient to sustain crops.

Pesticides can reach surface water through runoff from treated plantsand soil. Contamination of water by pesticides is widespread. Accordingto one comprehensive set of studies done by the U.S. Geological Survey(USGS) on major river basins across the country in the early tomid-1990s, more than 90 percent of water and fish samples from allstreams contained one, or more often, several pesticides. Pesticideswere found in all samples from major rivers with mixed agricultural andurban land use influences and 99 percent of samples of urban streams.The USGS also found that concentrations of insecticides in urban streamscommonly exceeded guidelines for protection of aquatic life.

Groundwater pollution due to pesticides is a worldwide problem.According to the USGS, at least 143 different pesticides and 21transformation products have been found in ground water, includingpesticides from every major chemical class. Over the past two decades,detections have been found in the ground water of more than 43 states.During one survey, 58 percent of drinking water samples drawn fromvarious hand pumps and wells were contaminated with Organo Chlorinepesticides at levels above EPA safety standards. Once ground water ispolluted with toxic chemicals, it may take many years for thecontamination to dissipate or be cleaned up. Cleanup may also be verycostly and complex, if not impossible.

Although pesticides are often considered a quick, easy and inexpensivesolution for controlling weeds, insects, bacteria and other pests, theiruse comes at a significant cost. Pesticides have contaminated almostevery part of the environment. Pesticide residues are found in soil andair, and in surface and ground water everywhere they are used.

Pesticide contamination poses significant risks to the environmentincluding non-target organisms ranging from beneficial soilmicroorganisms, to insects, plants, fish, birds, and humans.

It is with respect to this general technical environment that aspects ofthe present technology disclosed herein have been contemplated.Furthermore, although a general environment has been discussed, itshould be understood that the examples described herein should not belimited to the general environment identified in the background.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription section. This summary is not intended to identify keyfeatures or essential features of the claimed subject matter, nor is itintended to be used as an aid in determining the scope of the claimedsubject matter.

In general terms, this disclosure is directed to safely and effectivelyreducing pathogen levels on plants by exposing plants and their residentpathogens to gaseous ozone. This disclosure is also directed to safelyand effectively reducing fungus levels, including yeast and/or mold, onplants by exposing plants and their resident fungus to gaseous ozone.Various aspects are described in this disclosure, which include, but arenot limited to, the following aspects and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting examples and non-exhaustive examples are described withreference to the following figures.

FIG. 1 is a graph showing the average microbial counts from multipletrials after treatment of plant pathogens with gaseous ozone.

FIG. 2 is a graph showing microbial counts for individual trials aftertreatment of plant pathogens with gaseous ozone.

FIG. 3 is a graph showing E. Coli counts after treatment with gaseousozone at 100 ppm and 200 ppm compared with a control group.

FIG. 4 is a graph showing the average E. Coli counts from multipletrials after treatment with gaseous ozone at 100 ppm and 200 ppmcompared with a control group.

FIG. 5 is a graph showing Salmonella counts after treatment with gaseousozone at 100 ppm and 200 ppm compared with a control group.

FIG. 6 is a graph showing the average Salmonella counts from multipletrials after treatment with gaseous ozone at 100 ppm and 200 ppmcompared with a control group.

FIG. 7 is a graph showing Listeria counts after treatment with gaseousozone at 100 ppm and 200 ppm compared with a control group.

FIG. 8 is a graph showing the average Listeria counts from multipletrials after treatment with gaseous ozone at 100 ppm and 200 ppmcompared with a control group.

FIG. 9 is a graph showing Candida counts after treatment with gaseousozone at 100 ppm and 200 ppm compared with a control group.

FIG. 10 is a graph showing the average Candida counts from multipletrials after treatment with gaseous ozone at 100 ppm and 200 ppmcompared with a control group.

FIG. 11 is a graph and corresponding chart showing cannabinoid levelsfor a first strain of cannabis not treated with gaseous ozone.

FIG. 12 is a graph and corresponding chart showing cannabinoid levelsfor a cannabinoid test run on a first strain of cannabis after treatmentwith gaseous ozone.

FIG. 13 is a graph and corresponding chart showing cannabinoid levelsfor a second strain of cannabis not treated with gaseous ozone.

FIG. 14 is a graph and corresponding chart showing cannabinoid levelsfor a cannabinoid test run on a second strain of cannabis aftertreatment with gaseous ozone.

FIG. 15 is a graph showing terpene levels for a first strain of cannabisnot treated with gaseous ozone.

FIG. 16 is a graph showing terpene levels for a terpene test run on afirst strain of cannabis after treatment with gaseous ozone.

FIG. 17 is a graph showing terpene levels for a second strain ofcannabis not treated with gaseous ozone.

FIG. 18 is a graph showing terpene levels for a test run on a secondstrain of cannabis after treatment with gaseous ozone.

FIG. 19 is a simplified diagram of a distributed computing system inwhich aspects of the present invention may be practiced.

FIG. 20 illustrates one example of a suitable operating environment 1200in which aspects of the present invention may be implemented.

FIG. 21A illustrates a side profile of an embodiment of the pathogenreduction device 1908.

FIG. 21B illustrates a back profile of an embodiment of the pathogenreduction device 1908.

FIG. 21C illustrates an off center profile of an embodiment of thepathogen reduction device 1908.

FIG. 21D illustrates a top profile of an embodiment of the pathogenreduction device 1908.

FIG. 22 illustrates an embodiment of a tray used within the pathogenreduction device 1908 to hold plant while it is exposed to ozone.

DETAILED DESCRIPTION

Various embodiments will be described in detail with reference to thedrawings, wherein like reference numerals represent like parts andassemblies throughout the several views. Additionally, any examples setforth in this specification are not intended to be limiting and merelyset forth some of the many possible embodiments in which aspectsdisclosed herein may be practiced.

As used herein, the recited terms have the following meanings. All otherterms and phrases used in this specification, unless otherwisespecified, have their ordinary meanings as one of skill in the art wouldunderstand after review of this disclosure.

As used herein, “plant” can refer to any portion of a growing plant,including the roots, stems, stalks, leaves, branches, seeds, flowers,fruits, and the like.

As used herein, “pathogen” can refer to anything that causes disease orillness, but especially biological organisms such as bacteria, fungi,and viruses.

As used herein, “pesticide” refers to a composition or product thatkills or repels plant or seed pests, and may be broken into any numberof particular sub-groups including, but not limited to, acaricides,avicides, bactericides, fungicides, herbicides, insecticides, miticides,molluscicides, nematicides, piscicides, predacides, rodenticides andsilvicides. Pesticides may also include chemicals which are not normallyused as pest control agents, such as plant growth regulators, defoliantsand desiccants, or which are not directly toxic to pests, such asattractants and repellants. Some microbial pesticides may be bacteria,viruses and fungi that cause disease in given species of pests.Pesticides may be organic or inorganic. Pesticides applied to plantseeds may remain on the surface of the seed coat following application,or may absorb into the seed and translocate throughout the plant.

As used herein, “herbicide” refers to a composition or product thatkills or deters weed growth. One example of a herbicide includesglyphosate (i.e., RoundUp® herbicide).

As used herein, “insecticide” refers to a composition or product thatkills or repels insects. Examples of insecticides include Sevin(carbaryl), permethrin, and bacillus thruingiensis.

As used herein, “genetically modified plant” or “genetically modifiedorganism” refers to an organism whose genetic material has been alteredusing genetic engineering techniques such as recombinant DNA technology.

As used herein, “seed” refers to anything that can be sown to produce aplant. Seed can refer to an unfertilized plant ovule, a fertilized plantovule, and an embryonic plant. Seed can also refer to a whole or portionof a plant which is sown. For example, seed may refer to a whole orportion of a potato tuber.

As used herein, “applying” refers to bringing one or more componentsinto nearness or contact with another thing or component. Applying canrefer to contacting or administering.

As used herein, “fungus” refers to any member of the group of eukaryoticorganisms that includes microorganisms such as yeasts and molds.

Aspects of this disclosure relate to a method for reducing plantpathogens comprising: containing one or more of a seed, soil and a plantin a gaseous ozone chamber; concentrating gaseous ozone in the chamberand applying the concentrated gaseous ozone to the one or more of theseed, soil and the plant at a concentration of at least 100 ppm and atemperature in the range of 15° C. to 35° C. for at least 10 minutes.

Additional aspects relate to an ozone treatment system comprising: anoxygen concentrator configured to concentrate oxygen from ambient air;an ozone generator configured to adjust an ozone concentration in anozone chamber from 0.01 ppm to 500 ppm; one or more processors; and amemory coupled to the one or more processors, the memory for storinginstructions which, when executed by the one or more processors, causethe one or more processors to: determine a concentration of gaseousozone in an ozone chamber; determine an ambient temperature in the ozonechamber; adjust the concentration of gaseous ozone in the ozone chamberto a concentration in the range of 100 ppm to 500 ppm; adjust theambient temperature in the ozone chamber to a temperature in the rangeof 15° C. to 35° C.; and continuously monitor the concentration ofgaseous ozone and the ambient temperature in the ozone chamber andautomatically adjust the monitored concentration and temperature to apreset concentration and a preset temperature.

Other aspects relate to computer-readable media havingcomputer-executable instructions, that when executed by one or moreprocessors perform a method, the method comprising: receiving first datarelated to a concentration of gaseous ozone in an ozone chamber;receiving second data related to an ambient temperature in the ozonechamber; sending a request to a controller to adjust the concentrationof gaseous ozone in the ozone chamber to a concentration in the range of100 ppm to 500 ppm; and sending a request to the controller to adjustthe ambient temperature in the ozone chamber to a temperature in therange of 15° C. to 35° C.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the claimsattached hereto. Those skilled in the art will readily recognize variousmodifications and changes that may be made without following the exampleembodiments and applications illustrated and described herein, andwithout departing from the true spirit and scope of the claims.

FIG. 1 is a graph showing average microbial counts from three ozoneexposure trials for each of E. Coli, Salmonella, Listeria, and Candidainoculated Cannabis plants. Details regarding that study are as follows.

Cannabis plants were individually contaminated with Escherichia Coli,Salmonella enterica sv typhimurium, Listeria monocytogenes and Candidaalbicans then exposed to gaseous ozone for 20 minutes at 100 ppm and 200ppm. Significant kills of all bacterial pathogens were observed at 200ppm while a lesser kill rate was seen at 100 ppm except for theEscherichia coli which was observed to be significantly vulnerable toboth 100 ppm and 200 ppm ozone concentrations. The effect of ozonetreatment on Candida albicans were limited; however, testing at ozoneconcentrations higher than 200 ppm may provide markedly differentresults, as the results from the 200 ppm trials showed an averagereduced microbial count for Candida albicans when compared with thecontrol group (see FIG. 10).

Pure cultures of Escherichia coli and Listeria monocytogenes provided bythe American Type Culture Collection (ATCC) were grown in selectivemedia broths. The Candida albicans provided by ATCC was grown on potatodextrose agar, and after significant growth was achieved, the colonieswere transferred and suspended in Butterfield's buffer. Aftersignificant turbidity in each broth of pathogens was observed, they,along with Butterfield's buffer with the suspended Candida albicans,were used to inoculate individual Cannabis samples. This process createdfour individual populations of Cannabis with each population beingcontaminated with one of the four organisms grown. After inoculating thesamples they were allowed to dry overnight by placing them out in theopen at room temperature. Each population of Cannabis was then dividedinto smaller individual 2-4 gram samples which were used in the tests.One test consisted of placing nearly half of the smaller samples into anozone gas chamber for 20 minutes at an approximate ozone concentrationof 100 ppm. The remaining samples were set aside to be used as controls.

Three samples for each organism were tested at each of theconcentrations. The control samples were placed into the same chamberfor 20 minutes with an ozone concentration of 0 ppm. All samples werethen analyzed for their microbial counts by first generating serialdilution using Butterfield's buffer as the diluent for each sample thenindividually plating the resultant dilutions onto selective mediaappropriate for the organism being analyzed. After the requiredincubation temperatures and times were completed, the organisms on theplates were counted. For each set of dilutions of a given sample andstarting at the plate representing the lowest dilution ratio, the firstplate that was countable was counted and the result was recorded.

Testing occurred over two days. The first day involved testing onesample of Cannabis for each organism at an ozone concentration of 100ppm and another test of only Listeria at 50 ppm. Additional testinginvolving subsequent trials for each organism at ozone concentrations of100 ppm and 200 ppm were conducted on the second day of testing.

The ozone concentration in the ozone chamber was kept at a rangeapproximating the desired 100 ppm and 200 ppm concentrations. Forinstance, when the desired concentration was 100 ppm, the actual rangeof concentrations was 98 ppm to 106 ppm, and when the desiredconcentration was 200 ppm, the actual range of concentration was 198 ppmto 204 ppm. While the samples were being placed into the chamber ozonegas was lost. It took 3 minutes before the desired concentration of 100ppm was achieved for tests requiring that ozone concentration and 5minutes before the desired concentration of 200 ppm was achieved fortests requiring that ozone concentration.

The result of testing can be seen in Table 1 below. At an ozoneconcentration of 50 ppm, which only Listeria was involved with, therewas no statistically significant difference between the control and thetreated sample. At an ozone concentration of 100 ppm there was asignificant kill of all of the organisms involved in the test.

The results of the second day of testing can be seen in Table 2 below.There was a significant kill of all organisms at an ozone concentrationof 100 ppm when compared to the controls and at an ozone concentrationof 200 ppm the amount of kills increased when compared to the kills at100 ppm.

TABLE 1 This table shows the concentration range of gaseous ozonepresent during each test, the amount of time the organisms weresubjected to the ozone and the microbial counts after the treatmentsshown for the first day of testing. Treat- Target Ozone Actual Ozonement Microbial Concentration Concentration Time Count Microbe (ppm)Range (ppm) (min) (cfu/g) E. Coli Control 0 N/A 20 1.2 * 10¹¹ E. ColiTreated 100 95-102 20 2.8 * 10¹⁰ Salmonella 0 N/A 20 1.1 * 10¹¹ ControlSalmonella 100 95-102 20 1.7 * 10¹⁰ Treated Listeria Control 0 N/A 201.9 * 10⁷ Listeria Treated 100 95-102 20 4.8 * 10⁵ Listeria Control 0N/A 20 7.0 * 10² Listeria Treated 50 46-52  20 2.6 * 10² Mold Control 0N/A 20 1.4 * 10¹² Mold Treated 100 95-102 20 1.9 * 10¹¹

TABLE 2 This table shows the concentration range of the gaseous ozonepresent during each test, the amount of time the organisms weresubjected to the ozone and the microbial counts after treatments shownfor the second day of testing. Target Ozone Actual Ozone ConcentrationConcentration Treatment Time Microbial Count Microbe (ppm) Range (ppm)(min) (cfu/g) E. Coli Control 0 N/A 20 9.0 * 10⁴ E. Coli Trial 1 100 SeeTable 1 20 See Table 1 E. Coli Trial 2 100  98-106 20 BLD E. Coli Trial3 100  98-106 20 3.6 * 10¹ E. Coli Trial 1 200 198-204 20 BLD E. ColiTrial 2 200 198-204 20 2.3 * 10¹ E. Coli Trial 3 200 198-204 20 2.0 *10¹ Salmonella 0 N/A 20 4.5 * 10⁴ Control Salmonella Trial 1 100  98-10620 See Table 1 Salmonella Trial 2 100  98-106 20 1.1 * 10⁴ SalmonellaTrial 3 100 See Table 1 20 1.3 * 10⁴ Salmonella Trial 1 200 198-204 207.0 * 10² Salmonella Trial 2 200 198-204 20 2.2 * 10³ Salmonella Trial 3200 198-204 20 8.0 * 10³ Listeria Control 0 N/A 20 2.0 * 10⁴ ListeriaTrial 1 100 See Table 1 20 See Table 1 Listeria Trial 2 100  98-106 202.0 * 10³ Listeria Trial 3 100  98-106 20 1.0 * 10³ Listeria Trial 1 200198-204 20 3.0 * 10² Listeria Trial 2 200 198-204 20 3.1 * 10² ListeriaTrial 3 200 198-204 20 1.1 * 10² Candida Control 0 N/A 20 5.0 * 10⁵Candida Trial 1 100 See Table 1 20 See Table 1 Candida Trial 2 100 98-106 20 2.7 * 10⁵ Candida Trial 3 100  98-106 20 1.5 * 10⁴ CandidaTrial 1 200 198-204 20 1.0 * 10⁵ Candida Trial 2 200 198-204 20 8.3 *10⁴ Candida Trial 3 200 198-204 20 9.8 * 10⁴ “BLD” stands for below thelimit of detection.

This study shows that gaseous ozone is an effective antimicrobial stepwhen treating Cannabis samples contaminated with pathogens. It ispresumed that plant samples that would be treated for human consumptionwould not be near the level of contamination that this study generatedthrough the inoculation processes. Therefore the use of gaseous ozone asan antimicrobial step in production would assure that the level ofpathogen contamination after this step would be under the tolerancelevels set by safety guidelines.

The correlation between the reduction in pathogen levels and ozoneexposure depicted in the study can be extrapolated to many differentplant species. However, the study results are especially advantageous inshowing that a non-toxic means for reducing harmful pathogen levels toacceptable safety standards in the Cannabis plant is available.

Cannabis is grown under many different conditions, both indoors andoutdoors. As with all agricultural products, it is exposed to anextremely wide range of microorganisms.

The cannabinoids produced by the external glands of the Cannabis planthave well-documented antibacterial properties. Living Cannabis plants donot support high levels of bacterial growth, and pathogenic bacteria areunlikely to be associated with living Cannabis plants. There is alsoevidence for anti-fungal activity of certain cannabinoids, but fungalgrowth is not at all uncommon in Cannabis plants. Most of these mold andmildew species are plant pathogens, and not human ones; molds such asBotrytis cinerea may harm the Cannabis plant, but they are unlikely toharm humans.

Cannabis, as well as other plants such as herbs including mint and sage,citrus peel, some flowers and aromatic barks and woods, have distinctaromas produced by a terpenoid component in their essential oils. Thedistinct aromas that are given off by such plants are a product of whichterpenoids predominate for that plant. For example, in the case ofCannabis, terpenoids and cannabinoids are secreted inside the Cannabisplant's glandular trichomes, and they have a parent compound in common(geranyl pyrophosphate). More than 200 terpenoids have been identifiedin Cannabis. The most common and most studied include limonene, myrcene,alpha-pinene, linalool, beta-caryophyllene, caryophllene oxide,nerolidol and phytol. Because many consumers prize the distinct smellsproduced by aromatic plants, it is important that the plants maintainthose aromas even after being subjected to gaseous ozone treatment.

It has been determined that subjecting plants to concentrations of ozonebetween 1 ppm-1000 ppm for an amount of time in the range of 1 minute to48 hours produces plant products that maintain the distinct smellsproduced by those plants and their corresponding terpenes, even aftergaseous ozone treatment. Gaseous ozone treatment in these rangesprovides significant reduction or elimination of pathogens, pesticides,and fungus, including yeast and mold, while maintaining the distinctsmells intrinsic to aromatic plants. Further discussion regardingterpene content and gaseous ozone treatment is provided herein withrespect to the discussion of FIGS. 15-18.

In an embodiment, the concentration of ozone to which the target issubjected is between about 1 ppm and about 1000 ppm, about 1 ppm andabout 800 ppm, about 1 ppm and about 600 ppm, about 50 ppm and about 400ppm, about 50 ppm and about 300 ppm, about 100 ppm and about 300 ppm,about 150 ppm and about 250 ppm, or about 180 ppm and about 220 ppm. Insome embodiments, the concentration of ozone to which the target issubjected is greater than 20 ppm, greater than 50 ppm, greater than 75ppm, greater than 100 ppm, greater than 125 ppm, greater than 150 ppm,greater than 175 ppm, greater than 200 ppm, greater than 225 ppm,greater than 250 ppm, greater than 275 ppm, greater than 300 ppm,greater than 400 ppm, greater than 500 ppm, or greater than 600 ppm. Insome embodiments, the concentration of ozone to which the target issubjected is less than 700 ppm, less than 600 ppm, less than 500 ppm,less than 400 ppm, less than 350 ppm, less than 300 ppm, less than 275ppm, less than 250 ppm, less than 225 ppm, less than 200 ppm, less than175 ppm, less than 150 ppm, less than 125 ppm or less than 100 ppm. In apreferred embodiment, the concentration of ozone to which the target issubjected is between about 190 ppm to about 210 ppm.

Generally, it has been determined that the concentration of ozone usedis related to the exposure time necessary to achieve a desiredelimination of pathogens, pesticides, and fungus, including yeast andmold. For example, ozone levels as low as 1 ppm are effective at haltingthe growth process of mold or mildew if used over a longer period oftime. As a further example, if a plant is subjected to a concentrationof ozone of 200 ppm instead of 1 ppm, then less exposure time isnecessary to achieve a desired elimination of pathogens, pesticides, andfungus, including yeast and mold.

Importantly, the concentration of ozone and amount of time a plant isexposed to the ozone must account for several factors when determiningan effective concentration and exposure, including density of the plant(e.g., flower), the strain of the plant, and the surface area of plantthat is exposed to ozone. For example, a denser strain of cannabisflower (e.g., Indica or Indica dominant strain) may require moreexposure time to ozone than a less dense strain of flower (e.g., Sativaor Sativa dominant strain). In certain embodiments, if the strain offlower is too dense or too large (resulting in less surface area) thenthe plant may be modified (e.g., broken down into smaller pieces) toincrease the surface area and obtain more effective treatment. Ifworking with a cannabis plant, the exposures effect on cannabinoids(e.g., THC, CBGA, and THCA), terpene, trichomes, potency, flavorprofile, and weight should be considered. For example, using aconcentration of ozone (e.g., about 190 ppm to about 210 ppm) thateffectively reduces or eliminates pathogens, pesticides, and fungus,including yeast and mold with little to no negative effects on theplants cannabinoids (e.g., THC, CBGA, and THCA), terpene, trichomes,potency, flavor profile, and weight is desirable.

In an embodiment, the time a plant is exposed to ozone is about 1 minuteto about 48 hours, about 2 minutes to about 24 hours, about 3 minutes toabout 18 hours, about 4 minutes to about 12 hours, about 5 minutes toabout 6 hours, about 6 minutes to about 4 hours, about 7 minutes toabout 2 hours, about 8 minutes to about 1.5 hours, about 10 minutes toabout 1 hour, about 10 minutes to about 1 hour, about 12 minutes toabout 50 minutes, about 14 minutes to about 30 minutes, about 16 minutesto about 25 minutes. In some embodiments, the time a plant is exposed toozone is greater than 1 minute, greater than 5 minutes, greater than 10minutes, greater than 15 minutes, greater than 20 minutes, greater than30 minutes, greater than 45 minutes, greater than 1 hour, greater than 2hours, greater than 6 hours, greater than 12 hours, greater than 24hours or greater than 48 hours. In some embodiments, the time a plant isexposed to ozone is less than 48 hours, less than 24 hours, less than 12hours, less than 10 hours, less than 8 hours, less than 6 hours, lessthan 4 hours, less than 3 hours, less than 2 hours, less than 1.5 hours,less than 1 hour, less than 50 minutes, less than 40 minutes, less than30 minutes, less than 25 minutes, less than 20 minutes, less than 15minutes, less than 10 minutes, less than 5 minutes, or less than 2minutes. In a preferred embodiment, the time a plant is exposed to ozoneis about 20 minutes to about 60 minutes.

In an embodiment, a plant is exposed to ozone at room temperature (e.g.,59° F. to 77° F.). In an embodiment, a plant is exposed to ozone attemperatures between 40° F. and 100° F. In certain embodiments, a plantis exposed to ozone at temperatures greater than about 50° F., greaterthan about 60° F., greater than about 70° F., greater than about 80° F.,or greater than about 90° F. In certain embodiments, a plant is exposedto ozone at temperatures less than about 100° F., less than about 90°F., less than about 80° F., less than about 70° F., or less than about60° F.

It has been determined in certain embodiments that exposing a cannabisflower to a concentration of ozone of 200 ppm for between about 20minutes and 90 minutes can result in a reduction of 100,000 to 150,000CFUs per hour without any change to the potency, flavor profile,terpenes or weight of the flower.

In an embodiment, exposure of a plant to ozone results in less thanabout 50,000 CFUs on the treated plant following ozone exposure, lessthan about 40,000 CFUs, less than about 30,000 CFUs, less than about20,000 CFUs, less than about 10,000 CFUs, less than about 9,000 CFUs,less than about 8,000 CFUs, less than about 7,000 CFUs, less than about6,000 CFUs, less than about 5,000 CFUs, less than about 4,000 CFUs, lessthan about 3,000 CFUs, less than about 2,000 CFUs, less than about 1,000CFUs, less than about 500 CFUs, or no measurable CFUs.

Any danger to humans from consumption of pathogens associated with theCannabis plant would be due to a combination of factors across one ormore of the stages of: growth, processing, and use. Pathogens would haveto arrive on the plant during growing or processing, survive allprocessing and use steps, and then they—or their toxins—would have to betransferred to a human host in a way that allows them to cause disease.

Although Cannabis, unlike many other plants, has inherent antibacterialproperties, is dried well and is usually then heated during processingor use, microbial threats still exist. For example, detection ofsignificant levels of E. Coli are strong evidence of problems duringgrowing or processing, including contaminated soil or water, or improperhandling. E. Coli is accepted to be the optimal indicator organism forthe identification of possible fecal contamination. Were pathogenicbacteria such as E. Coli or Salmonella to be present, they would likelyhave arrived through this type of pathway, therefore samples for E. Coliare both higher risk and indicative of general production problems thatneed to be addressed.

Turning back to FIG. 1, test results for the study described above areprovided. The X-axis of graph 100 is divided into average test resultsfor gaseous ozone treatment of Cannabis inoculated with E. Coli,Salmonella, Listeria and Candida compared against control (i.e.,inoculated Cannabis not treated with ozone). The Y-axis of graph 100depicts averages for the test results, in log values of colony-formingunits per gram (CFU/gram), for the study. Log values for averageCFU/gram for E. Coli are shown at 102. Log values for average CFU/gramfor Salmonella are shown at 104. Log values for average CFU/gram forListeria are shown at 106 and log values for average CFU/gram forCandida are shown at 108.

Moving to FIG. 2, test results for the study described above aredepicted in graph 200. Microbial counts for the individual trialsconducted during that study are shown for E. Coli, Salmonella, Listeriaand Candida against control.

FIG. 3 is a graph 300 showing E. Coli counts after treatment withgaseous ozone compared with a control group. Results for the controlgroup are shown at 302, results for the group treated at an ozoneconcentration of 100 ppm are shown at 304 and results for the grouptreated at an ozone concentration of 200 ppm are shown at 306.

Turning to FIG. 4 average E. Coli counts after treatment with gaseousozone compared with a control group are depicted in graph 400. Resultsfor the control group are shown at 402, average results for the grouptreated at an ozone concentration of 100 ppm are shown at 404 andaverage results for the group treated at an ozone concentration of 200ppm are shown at 406.

FIG. 5 is a graph 500 showing Salmonella counts after treatment withgaseous ozone compared with a control group. Results for the controlgroup are shown at 502, results for the group treated at an ozoneconcentration of 100 ppm are shown at 504 and results for the grouptreated at an ozone concentration of 200 ppm are shown at 506.

FIG. 6 shows a graph 600 depicting average Salmonella counts aftertreatment with gaseous ozone compared with a control group. Results forthe control group are shown at 602, average results for the grouptreated at an ozone concentration of 100 ppm are shown at 604 andaverage results for the group treated at an ozone concentration of 200ppm are shown at 606.

FIG. 7 is a graph 700 showing Listeria counts after treatment withgaseous ozone compared with a control group. Results for the controlgroup are shown at 702, results for the group treated at an ozoneconcentration of 100 ppm are shown at 704 and results for the grouptreated at an ozone concentration of 200 ppm are shown at 706.

FIG. 8 shows a graph 800 depicting average Listeria counts aftertreatment with gaseous ozone compared with a control group. Results forthe control group are shown at 802, average results for the grouptreated at an ozone concentration of 100 ppm are shown at 804 andaverage results for the group treated at an ozone concentration of 200ppm are shown at 806.

FIG. 9 is a graph 900 showing Candida counts after treatment withgaseous ozone compared with a control group. Results for the controlgroup are shown at 902, results for the group treated at an ozoneconcentration of 100 ppm are shown at 904 and results for the grouptreated at an ozone concentration of 200 ppm are shown at 906.

FIG. 10 shows a graph 1000 depicting average Candida counts aftertreatment with gaseous ozone compared with a control group. Results forthe control group are shown at 1002, average results for the grouptreated at an ozone concentration of 100 ppm are shown at 1004 andaverage results for the group treated at an ozone concentration of 200are shown at 1006.

FIG. 11 and FIG. 12 depict graphs 1102 and 1202 showing cannabinoidlevels for a first strain of cannabis. The results shown in relation toFIG. 11 are for a control sample (i.e., a cannabis sample that has notbeen subjected to gaseous ozone). The results shown in relation to FIG.12 are for a sample that was treated with gaseous ozone according to themethods described herein. Also shown are detailed charts 1104 and 1204depicting more precise percentages of the cannabinoid levels for thattested strain, including levels for THC-A, CBD, CBD-A, CBN, CBG, CBG-A,CBC and CHCV. THC-A and CBD-A are the “inactive” acidic forms of the THCand CBD molecules, which convert THC and CBD given time and/or heat.THC-A and CBD-A counts, as reflected in the graphs 1102 and 1202 and thecharts 1104 and 1204 have been adjusted to account for the greaterweight of the acidic molecules.

FIG. 13 and FIG. 14 depict graphs 1302 and 1402 showing cannabinoidlevels for a second strain of cannabis. The results in relation to FIG.13 are for a control sample (i.e., a cannabis sample that has not beensubjected to gaseous ozone). The results shown in relation to FIG. 14are for a sample that was treated with gaseous ozone according to themethods described herein. Also shown are detailed charts 1304 and 1404showing more precise percentages of the cannabinoid levels for thatsecond tested strain, including levels for THC-A, CBD, CBD-A, CBN, CBG,CBG-A, CBC and CHCV. THC-A and CBD-A counts, as reflected in the graphs1302 and 1402 and the charts 1304 and 1404 have been adjusted to accountfor the greater weight of the acidic molecules.

The results from the tests as shown in FIGS. 11-14 show that cannabinoidlevels are not adversely affected by treatment with gaseous ozoneaccording to methods described herein. Rather, total THC for the testedsamples for both the control samples and samples treated with gaseousozone according to the methods described herein were significantlysimilar. That is, cannabis samples treated with gaseous ozone maintaincannabinoid concentrations similar to cannabis samples that have notbeen treated with gaseous ozone.

FIG. 15 and FIG. 16 are graphs 1500 and 1600 depicting various levels ofterpenes 1502 and 1602 found in a first strain of cannabis. The Y axisof the graphs 1500 and 1600 represent terpene concentration percent byweight. The results shown in relation to FIG. 15 are for a controlsample (i.e., a cannabis sample that has not been subjected to gaseousozone). The results shown in relation to FIG. 16 are for a sample thatwas treated with gaseous ozone according to the methods describedherein. Also shown in the graphs 1500 and 1600 is the total amount ofterpenes 1504 and 1604 found in the first strain for both the controlsamples (FIG. 15) and the treated samples (FIG. 16).

FIG. 17 and FIG. 18 are graphs 1700 and 1800 depicting various levels ofterpenes 1702 and 1802 found in a second strain of cannabis. The Y axisof the graphs 1700 and 1800 represent terpene concentration percent byweight. The results shown in relation to FIG. 17 are for a controlsample (i.e., a cannabis sample that has not been subjected to gaseousozone). The results shown in relation to FIG. 18 are for a sample thatwas treated with gaseous ozone according to the methods describedherein. Also shown in graphs 1700 and 1800 is the total amount ofterpenes 1704 and 1708 found in the second strain for both controlsamples (FIG. 17) and treated samples (FIG. 18).

The results of the tests as shown in FIGS. 15-18 show that terpenelevels are not adversely affected by treatment with gaseous ozoneaccording to methods described herein. Rather, total terpene levels forthe tested samples for both the control samples and samples treated withgaseous ozone were significantly similar. That is, cannabis samplestreated with gaseous ozone maintain terpene concentrations similar tocannabis samples that have not been treated with gaseous ozone.

Terpene Testing Methods: A testing methodology known as HeadspaceGas-Chromatography with Flame Ionization Detection, or headspace GC-FIDwas used. This method is widely used in the environmental andpharmaceutical industries to analyze for product or environmentalcontamination. For each test, a small sample of cannabis is used. Thesample is heated in an airtight vial to vaporize the residual solvents,sample the headspace in the vial and inject the headspace sample into agas chromatograph for chemical analysis. In analyzing sample headspace,various matrix interferences were screened from the concentrate. Theterpene content for the samples may represent lower than expectedresults as no correction for moisture content was performed. As such,some terpenes may have evaporated upon drying giving lower than expectedterpene results.

FIG. 19 is a simplified diagram of a distributed computing system 1914in which aspects of the present invention may be practiced. According toexamples, any of computing devices 1902A (a modem), 1902B (a laptopcomputer), 1902C (a tablet), 1902D (a personal computer), 1902E (a smartphone), and 1902F (a server) may be used to send, receive and evaluatesignals from pathogen reduction device 1908 via one or more networkservers 1906 and a network 1920. Such signals may include data relatedto ozone concentration, temperature and time of exposure, for example.

According to some aspects pathogen reduction device 1908 may be astationary or fixed device. According to other aspects pathogenreduction device 1908 may be a mobile device. For example, pathogenreduction device 1908 may stand on a plurality of wheels for moving thedevice from one place to another. The wheels may be fixed to the deviceor they may be readily removed and put back on, by for example, a popout mechanism. According to an embodiment pathogen reduction device 1908may have the following dimensions: a length of 4 feet, 4 inches; a widthof 2 feet, 0 inches; and a height of 5 feet, 2 inches. With wheelsattached the height of the pathogen reduction device may be 5 feet, 5inches.

According to additional examples pathogen reduction device 1908 maycontain a plurality of racks within an ozone chamber. The racks may bepositioned suitably for treating plants on each rack level in thepathogen reduction device 1908. For example, the racks may be positionedat 6 inch vertical intervals within the pathogen reduction device 1908.The racks may be made of metal sliders and a metal mesh shelf toeffectively ozonate a plant. They may also include a lip around theshelf to prevent loss of a treated plant. For example, the shelves mayinclude a 3 inch lip such that treated plant product is not lost. Inaccordance with these examples it should be appreciated that such aracking system allows for the processing (i.e., gaseous ozone treatment)of approximately 33-44 pounds of plant product every 20 minutes.

Pathogen reduction device 1908 may comprise one or more of a controller,an ozone system (including an ozone generator and an ozone chamber) andan oxygen concentrator.

Pathogen reduction device 1908 according to certain embodiments mayinclude safety mechanisms including but not limited to a destructor forventing gaseous ozone, providing a mechanism for immediately degradingozone back to O₂, a leak sensor in communicative contact with an alarmdisplay and a safety interlock. According to aspects, one or more ofthese safety mechanisms may be employed as part of pathogen reductiondevice 1908 as well as distributed computing system 1914.

A controller as described herein in association with the pathogenreduction device 1908 may control and operate each component within thepathogen reduction device 1908 including the ozone chamber. Thecontroller may comprise one or more processors and a memory coupled tothe one or more processors. The memory may store instructions that whenexecuted by the one or more processors cause the one or more processorsto implement one or more steps, including: determining a concentrationof gaseous ozone in an ozone chamber; adjusting the concentration ofgaseous ozone in the ozone chamber; adjusting the ambient temperature inthe ozone chamber; continuously monitoring the concentration of gaseousozone and the ambient temperature in the ozone chamber and automaticallyadjusting the monitored concentration and temperature to a presetconcentration and preset temperature.

The controller may also include a graphical user interface for touchscreen operation and system interaction. Integrated sensors may beconfigured to monitor conditions in the pathogen reduction device 1908so that proper action can be taken to reduce pathogen levels associatedwith plants being treated in the pathogen reduction device 1908. Forexample, integrated sensors may provide, via a graphical user interfaceon the pathogen reduction device or a graphical user interface oncomputing devices 1902A-F, an indication that an ozone leak hasoccurred. The controller may be further configured to shut down one ormore of the elements described in the pathogen reduction methods andsystems described herein to protect the various components of thepathogen reduction device 1908. The controller may also be configured tosend a signal to one or more of computing devices 1902A-F if a sensorhas failed such that remedial action can be taken.

FIG. 20 illustrates one example of a suitable operating environment 2000in which one or more of the present embodiments may be implemented. FIG.20 provides only one example of a suitable operating environment and isnot intended to suggest any limitation as to the scope of use orfunctionality. Other well-known computing systems, environments, and/orconfigurations that may be suitable for use include, but are not limitedto, personal computers, server computers, hand-held or laptop devices,multiprocessor systems, microprocessor-based systems, programmableconsumer electronics such as smart phones, network PCs, minicomputers,mainframe computers, distributed computing environments that include anyof the above systems or devices, and the like.

In its most basic configuration, operating environment 2000 typicallyincludes at least one processing unit 2002 and memory 2004. Depending onthe exact configuration and type of computing device, memory 2004(storing, among other things, reputation information, categoryinformation, cached entries, instructions to perform the methodsdisclosed herein, etc.) may be volatile (such as RAM), non-volatile(such as ROM, flash memory, etc.), or some combination of the two. Thismost basic configuration is illustrated in FIG. 20 by dashed line 2006.Further, environment 2000 may also include storage devices (removable,2008, and/or non-removable, 2010) including, but not limited to,magnetic or optical disks or tape. Similarly, environment 2000 may alsohave input device(s) 2014 such as keyboard, mouse, pen, voice input,etc. and/or output device(s) 2016 such as a display, speakers, printer,etc. Also included in the environment may be one or more communicationconnections, 2012, such as LAN, WAN, point to point, etc.

Operating environment 2000 typically includes at least some form ofcomputer readable media. Computer readable media can be any availablemedia that can be accessed by processing unit 2002 or other devicescomprising the operating environment.

By way of example, and not limitation, computer readable media maycomprise computer storage media and communication media. Computerstorage media includes volatile and nonvolatile, removable andnon-removable media implemented in any method or technology for storageof information such as computer readable instructions, data structures,program modules or other data. Computer storage media includes, RAM,ROM, EEPROM, flash memory or other memory technology, CD-ROM, digitalversatile disks (DVD) or other optical storage, magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage devices,or any other tangible medium which can be used to store the desiredinformation. Computer storage media does not include communicationmedia.

Communication media embodies computer readable instructions, datastructures, program modules, or other data in a modulated data signalsuch as a carrier wave or other transport mechanism and includes anyinformation delivery media. The term “modulated data signal” means asignal that has one or more of its characteristics set or changed insuch a manner as to encode information in the signal. By way of example,and not limitation, communication media includes wired media such as awired network or direct-wired connection, and wireless media such asacoustic, RF, infrared and other wireless media. Combinations of the anyof the above should also be included within the scope of computerreadable media.

The operating environment 2000 may be a single computer operating in anetworked environment using logical connections to one or more remotecomputers. The remote computer may be a personal computer, a server, arouter, a network PC, a peer device or other common network node, andtypically includes many or all of the elements described above as wellas others not so mentioned. The logical connections may include anymethod supported by available communications media. Such networkingenvironments are commonplace in offices, enterprise-wide computernetworks, intranets and the Internet.

Aspects described herein may be employed using software, hardware, or acombination of software and hardware to implement and perform thesystems and methods disclosed herein. Although specific devices havebeen recited throughout the disclosure as performing specific functions,one of skill in the art will appreciate that these devices are providedfor illustrative purposes, and other devices may be employed to performthe functionality disclosed herein without departing from the scope ofthe disclosure.

It has been determined that an embodiment of a disclosed pathogenreduction device can ensure a constant level of ozone gas (e.g., 200ppm) throughout the exposure time. In addition, an embodiment of adisclosed pathogen reduction device is capable of treating a smallsample (e.g., less than 10 grams of flower) or large sample (e.g.,enough sample to completely fill all the racks of the pathogen reductiondevice, which varies depending on device size but can be large, e.g., 50lbs) without negatively affecting the effectiveness of the ozonetreatment.

The ability of ozone to reduce pesticide residue using an embodiment ofa disclosed pathogen reduction device was evaluated. In this series ofexperiments, Myclobutanil and Bifenizate were purchased in liquidformulations (Myclobutanil at 19.7% from Dow AgroSciences as Eagle® 20EWand Bifenazate at 22.6% from Chemtura Corporation as Floramite® SC). Thepesticides were sprayed on homogenized hemp flower and air dried. Driedhemp flower with pesticide was then exposed to treatment with 200 ppm ofozone for 20 minutes. Non pesticide treated hemp, pesticide treatedhemp, and pesticide treated hemp that was exposed to ozone were analyzedby spectrometry using AOAC official method 2007.01. The results aredisclosed below in Table 3 were Matrix Blank is non pesticide treatedhemp (negative control), Control is pesticide treated hemp that was notexposed to ozone, and Sample 1, Sample 2, and Sample 3 are three samplesof pesticide treated hemp that was exposed to ozone at 200 ppm for 20minutes.

Ozone treatment of hemp contaminated with Myclobutanil or Bifenizate waseffective. For example, exposing hemp contaminated with Myclobutanil for20 minutes at 200 ppm ozone resulted in an average reduction of 0.18 ppmMyclobutanil across the three samples tested. Similarly, exposing hempcontaminated with Bifenizate for 20 minutes at 200 ppm ozone resulted inan average reduction of 0.1 ppm Bifenizate across the three samplestested.

TABLE 3 This table discloses the ability of ozone treatment toeffectively reduce Myclobutanil or Bifenizate. Matrix Blank ControlSample 1 Sample 2 Sample 3 Compound (mg/kg) (mg/kg) (mg/kg) (mg/kg)(mg/kg) Myclobutanil LOD 0.5 0.39 0.28 0.28 Bifenazate LOD 0.34 0.280.21 0.22 “LOD” stands for below the limit of detection.

The ability of ozone to reduce fungus using an embodiment of a disclosedpathogen reduction device was also evaluated. In this series ofexperiments, homogenized cannabis flower with a total yeast and moldbio-burden exceeding 10,000 CFU/g was exposed to ozone using anembodiment of a disclosed pathogen reduction device. 20 gram samples ofthe cannabis flower were separated into two groups (10 grams a group)and placed on different racks within the pathogen reduction device. One10 gram sample was placed on an industrial grade aluminum rack with airholes in order to maximize ozone flow and increase the surface area ofthe flower that would be exposed to ozone. A second 10 gram sample wasplace directly onto a rack screen (aluminum foil with holes pocked intoit); however, because some of the homogenized cannabis flower was finerthan the rack screen, some flower from the second 10 gram sample passedthrough the rack screen and landed on the lowest mesh screen in thedevice, thereby, creating a third sample for testing.

20 gram samples as described above were exposed to ozone for 0 minutes(control), 20 minutes, 30 minutes, 45 minutes, and 60 minutes at anozone concentration of 200 ppm. Six 1 gram samples from each treatmenttime were processed and evaluated using a 3M® Petrifilm® Rapid Yeast andMold Count Plate and accompanying Product Instructions. The ozonetreated samples were homogenized, diluted with buffer (distilled water),and then 1 milliliter suspension samples were dispensed onto 3M®Petrifilm® Rapid Yeast and Mold Count Plates. The plates were incubatedat 27° C. for greater than 60 hours in order to quantify the total yeastand mold remaining on the cannabis flower following exposure to ozone.Three of the six 1 gram samples were taken from the flower placed on theindustrial grade aluminum rack as detailed above. Two of the 1 gramsamples were taken from the flower placed directly on the screen. Thefinal 1 gram sample was collected from the lowest mesh screen in thedevice where the finest flower collected after falling through the rackscreen.

As detailed in the following Table 4 and Table 5, a 92% decrease intotal yeast and mold bio-burden was observed after treatment with ozone,with Colony Forming Units (CFUs)/gram dropping from an average of 82,000CFU/g to an average of 7,000 CFR/g after treatment with ozone for 60minutes. The most significant reduction in CFU/g was observed in thefirst 20 minutes of exposure to ozone. After the first 20 minutes and upuntil 45 minutes, there was only an additional 1.1% reduction in CFU/g.However, between a 45 minute and 60 minute exposure to ozone, there wasanother 14.1% reduction in CFU/g. Notably, there was a reduction inyeast and mold across all 6 samples from roughly 10⁵ CFU/g to below10,000 CFU/g following treatment with ozone in an embodiment of adisclosed pathogen reduction device.

TABLE 4 Average CFU/g of yeast and mold remaining on the tested cannabisflower following exposure to ozone for varying periods of time. Sample(exposure time in Average CFU/g minutes) (all samples tested)  0 minutes68,000 20 minutes 18,000 30 minutes 17,000 45 minutes 16,000 60 minutes6,000

Table 4 reveals that fungi are strongly inhibited by ozone exposure.Notably, graphing the results of Table 4 (exposure time versus averageCFU/g) allows an estimation of treatment time for sample with higherlevels of fungal contamination than tested in Table 4. The data tosupport Table 4 is disclosed in Table 5.

TABLE 5 Results from experiments described above and presented in Table4. Sample (6 samples for each exposure Location in Pathogen ReductionCFU/g CFU/g in minutes) Device (Test 1) (Test 2) T0-1 Lowest mesh screen77,000 61,000 T0-2 Rack screen 30,000 28,000 T0-3 Rack screen 50,00077,000 T0-4 Industrial grade aluminum rack 25,000 53,000 T0-5 Industrialgrade aluminum rack 60,000 140,000 T0-6 Industrial grade aluminum rack82,000 130,000 T20-1 Lowest mesh screen 18,000 24,000 T20-2 Rack screen15,000 16,000 T20-3 Rack screen 15,000 17,000 T20-4 Industrial gradealuminum rack 19,000 23,000 T20-5 Industrial grade aluminum rack 12,00018,000 T20-6 Industrial grade aluminum rack 21,000 21,000 T30-1 Lowestmesh screen 16,000 20,000 T30-2 Rack screen 18,000 30,000 T30-3 Rackscreen 13,000 16,000 T30-4 Industrial grade aluminum rack 16,000 15,000T30-5 Industrial grade aluminum rack 11,000 14,000 T30-6 Industrialgrade aluminum rack 20,000 20,000 T45-1 Lowest mesh screen 7,100 11,00045-2 Rack screen 13,000 15,000 T45-3 Rack screen 11,000 12,000 T45-4Industrial grade aluminum rack 21,000 48,000 T45-5 Industrial gradealuminum rack 16,000 17,000 T45-6 Industrial grade aluminum rack 8,00012,000 T60-1 Lowest mesh screen 7,400 9,000 T60-2 Rack screen 6,00010,000 T60-3 Rack screen 7,800 6,300 T60-4 Industrial grade aluminumrack 770 3,100 T60-5 Industrial grade aluminum rack 4,300 4,400 T60-6Industrial grade aluminum rack 6,300 7,100 T0-1 represents sample 1 withan ozone exposure time of 0 minutes. T20-1 represents sample 1 with anozone exposure time of 20 minutes. T30-1 represents sample 1 with anozone exposure time of 30 minutes. T45-1 represents sample 1 with anozone exposure time of 45 minutes. T60-1 represents sample 1 with anozone exposure time of 60 minutes.

Some additional ozone exposure tests were performed with cannabis floweror trim on a variety of commercially available strains using anembodiment of a disclosed pathogen reduction device. In each experiment(disclosed in Table 6) a reduction of mold and yeast CFU/g was measured(as disclosed above using 3M® Petrifilm® Rapid Yeast and Mold CountPlate and Product Instruction) after treating different weights ofcannabis with an ozone concentration of 200 ppm for between 20 and 60minutes.

TABLE 6 Results of ozone treatment of cannabis flower or trim to reducemold and yeast. CFUs were measure before treatment (Initial CFU) andafter treatment (Ending CUF) with ozone. Total Weight OzoneConcentration Type of Product Strain Type Initial CFU Ending CFU Run(lbs) (ppm) Length of Time (min.) Flower Hybrid/Sativa 100,0005,400-5,500 1 lb  200 20:00 Flower Hybrid/Indica 51,750 30,000-32,000 6lbs 200 25:00 Trim Hybrid/Indica 17,000   800-11,000 6 lbs 200 25:00Flower Hybrid/Indica 2,200 220 1 lb  200 45:00 Flower Indica 320,00091,000 3 lbs 200 60:00 Trim Hybrid/Sativa 50,000 3,000 3 lbs 200 60:00Flower Hybrid/Indica 110,000 3,000 NA 200 60:00 Flower Hybrid 77,0004500 5 lbs 200 60:00

While ozone exposure can successfully reduce pesticides and fungus oncannabis flower, it has little to no effect on the terpenes of acannabis flower. Cannabinoid and terpene content of cannabis flower wasmeasured before exposure to 200 ppm of ozone in an embodiment of adisclosed pathogen reduction device and after a 60 minute exposure. Theresults are disclosed in Table 7 by the different cannabinoids andterpenes measured.

TABLE 7 A 60 minute exposure of cannabis flower to ozone had minimal tono effect on the cannabinoid and terpene content. % Before % AfterCannabinoid Ozone Exposure Ozone Exposure % Change or Terpene (0minutes) (60 minutes) (decrease) % THC 0.749 0.680 9.2 % THCA 16.9 15.77.1 % CBDA 0.037 0.035 5.4 % CBCA 0.105 0.102 0.28 % CBGA 0.638 0.55213.4 % CBNA 0.046 0.0458 0.04 % THCVA 0.077 0.076 1.2 Total Terpene %2.11 1.94 8.1

Although specific examples were described herein, the scope of thetechnology is not limited to those specific examples. One skilled in theart will recognize other aspects, examples or improvements that arewithin the scope and spirit of the present technology. Therefore, thespecific structure, acts, or media are disclosed only as illustrativeexamples according to the disclosure.

1. A method for reducing the microbial count of plant pathogens in a sample of cannabis flower, comprising the steps of: a. obtaining a pathogen reduction device comprising, (i) a controller; and (ii) an ozone system including an ozone generator, an ozone chamber and an oxygen concentrator; b. placing a sample of cannabis flower comprising a plant pathogen in the ozone chamber; c. providing a preset concentration of ozone gas in the ozone chamber; and d. monitoring the concentration of ozone gas in the ozone chamber and automatically adjusting the concentration of ozone gas to a preset ozone concentration of from about 50 ppm to about 400 ppm, wherein when the sample of cannabis flower is exposed to ozone gas in the ozone chamber for a time period of from 1 minute to 48 hours the microbial count of plant pathogens in the sample of cannabis flower is reduced.
 2. The method according to claim 1, wherein the oxygen concentrator is configured to concentrate oxygen from ambient air.
 3. The method according to claim 2, wherein the sample of cannabis flower comprising a plant pathogen is exposed to ozone gas for a preset time period selected from the group consisting of greater than 30 minutes, greater than 45 minutes, greater than 1 hour, greater than 2 hours, greater than 6 hours, greater than 12 hours, greater than 24 hours, and less than 48 hours.
 4. The method according to claim 2, wherein the sample of cannabis flower comprising a plant pathogen is exposed to ozone gas for a preset time period selected from the group consisting of 20 minutes to 90 minutes, 30 minutes to 480 minutes, 60 minutes to 130 minutes, 180 minutes to 360 minutes, 12 hours to 16 hours, greater than 12 hours, less than 24 hours, and less than 48 hours.
 5. The method according to claim 4, wherein the preset concentration of gaseous ozone in the ozone chamber is maintained in the range of about 100 ppm to 300 ppm or about 150 ppm to 250 ppm.
 6. The method according to claim 5, wherein the preset concentration of gaseous ozone in the ozone chamber is maintained in the range of about 200 to 250 ppm.
 7. The method according to claim 4, wherein the sample is homogenized cannabis flower.
 8. The method according to claim 4, wherein the sample of cannabis flower comprising a plant pathogen is placed in the ozone chamber for 20 minutes at an ozone concentration of about 200 ppm, and the method is effective to reduce the amount of E. coli, Salmonella, or Listeria in the sample by greater than 90%.
 9. The method according to claim 6, wherein the microbial count is total yeast and mold colony forming units (CFU), the total CFU in the sample of cannabis flower is approximately 10⁵ CFU/g, and after exposure to an ozone concentration of 200 ppm in the ozone chamber for 20 to 90 minutes, the method is effective to reduce the CFU to below 10,000 CFU/g.
 10. The method according to claim 6, wherein the microbial count is total yeast and mold CFU, and the total CFU in the sample of cannabis flower is reduced by greater than 90% following exposure to an ozone concentration of 200 ppm in the ozone chamber for 60 minutes.
 11. The method according to claim 6, wherein the microbial count is total yeast and mold CFU, and the method is effective to reduce the CFU in a sample of cannabis flower by 100,000 CFU, following exposure to an ozone concentration of 200 ppm in the ozone chamber for about 20 to 90 minutes.
 12. The method according to claim 6, wherein the microbial count is total yeast and mold CFU, and the method is effective to reduce the microbial count by 50,000 CFU, following exposure to an ozone concentration of 200 ppm in the ozone chamber for about 20 to 90 minutes.
 13. The method according to claim 6, wherein the THC and THCA levels in a sample of cannabis flower comprising a plant pathogen are substantially the same following exposure to an ozone concentration of about 200 ppm in the ozone chamber for about 20 to 90 minutes.
 14. The method according to claim 6, wherein the terpene levels in a sample of cannabis flower comprising a plant pathogen are substantially the same following exposure to an ozone concentration of about 200 ppm in the ozone chamber for about 20 to 90 minutes. 